Abstract of Parental Grant GM124186 Elucidation of the mechanisms by which endocytic trafficking regulates signal transduction processes remains to be profoundly important for understanding how diverse stimuli propagate signals from the cell surface through the conserved cytosolic machinery and to the nucleus leading to a stimulus- and context-specific signaling outcome. Epidermal growth factor (EGF) receptor (EGFR), a prototypic receptor tyrosine kinase, has been the major experimental model to study spatiotemporal regulation of signaling networks. EGFR plays an essential role in mammalian development and tissue homeostasis in the adult, and is involved in human pathogenesis, in particular, cancer. However, while the main constituents of the EGFR signaling network are known, how they coordinately function during EGFR endocytosis and subsequent targeting of receptors for degradation to endosomes to ensure proper intensity and duration of signaling processes is for the large part unknown. Addressing this fundamental question has now become possible owing to the availability of new cutting- edge methodologies. Using time-resolved quantitative mass-spectrometry of cellular phosphoproteomes we found that after the majority of active EGFRs are internalized into endosomes, phosphorylation of a multitude of proteins, known to be involved in the regulation of signaling to growth, survival, cell motility and adhesion, is maintained by EGFR. Therefore, we hypothesize that the pathways involving these putative signaling effectors of EGFR operate in endosomes through the sustained activity of endosomal EGFR. We further hypothesize that by maintaining the activity along some signaling pathways while down-regulating other pathways, EGFR endocytosis shapes the overall functional outcome of EGFR signaling. To test these hypothesis in the physiological experimental system (cells that are growth-dependent on EGFR) we will: 1) examine whether putative substrates and signaling effectors of endocytosed EGFR identified by the phosphoproteomic analysis are located in EGFR-containing endosomes by labeling endogenous effectors with fluorescent proteins by gene-editing and dissecting their time-dependent localization dynamics using multi- dimensional fluorescence microscopy imaging; and 2) examine whether endocytosis and localization in endosomes control downstream signaling activity of putative phosphorylation substrates and effectors of endosomal EGFR, and define the mechanisms of this regulation. Combination of the high-throughput method of labeling endogenous proteins by gene-editing with various methods of quantitative live-cell optical imaging at all levels of resolution will allow us to apply a systems biology approach to untangling the entire endosomal signaling program.